Method of inhibiting the activity of human immunodeficiency virus (HIV) in vivo

ABSTRACT

A method for inhibiting the activity of human immunodeficiency virus (HIV) in vivo comprises administering to a human host an antimalarial drug, which is capable of exhibiting a protective effect, a curative effect, or of preventing transmission of malaria in humans. The antimalarial drug is selected from the group consisting of 
     (a) alkaloids; 
     (b) 9-amino-acridines; 
     (c) 4-aminoquinolines; 
     (d) 8-aminoquinolines; 
     (e) biguanides; 
     (f) dihyrofolate reductase inhibitors; 
     (g) sulfones; 
     (h) sulfonamides; 
     (i) mefloquine; 
     (i) halofantrine; 
     (k) hydroxyanilino-benzo-naphtyridines; and 
     (l) sesquiterpene lactones. 
     The antimalarial drug is administered to the human in an amount sufficient to prevent or at least inhibit infection of T lymphocytes by HIV in vivo or to prevent or at least inhibit replication of HIV in vivo.

This application is a continuation of U.S. Ser. No. 07/796,244, filedNov. 25, 1991 and now abandoned, which is a divisional of applicationSer. No. 690,314, filed Apr. 25, 1991 and now U.S. Pat. No. 5,153,202;which is a continuation application of Ser. No. 560,467, filed Jul. 27,1990 and now abandoned; which is a continuation application of Ser. No.213,811, filed Jun. 30, 1988 and now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the use of anti-malarial drugs for inhibitinginfection of susceptible cells by human immunodeficiency virus (HIV).This invention also relates to a method of inhibiting proliferation ofHIV.

Acquired immune deficiency syndrome (AIDS) is a condition which is nowof major importance in North America, Europe, and Central Africa. Thecasual agent of AIDS is believed to be a retrovirus. Recent estimatessuggest that approximately 1.5 million Americans may have been exposedto the AIDS virus. The individuals affected show severeimmunosuppression, which may be followed by the onset of degenerativeand even fatal diseases.

The isolation and characterization of the first AIDS retrovirus, knownas LAV, was described in a paper by F. Barre-Sinoussi, et al. Science,220:868-871 (1983). The use of some extracts of this virus and some ofits proteins to detect antibodies against the virus is described in U.S.Pat. No. 4,708,818 issued to Dr. Luc Montagnier, et al.

Several isolates of the AIDS retrovirus were subsequently reported bydifferent investigators and-the isolates were referred to in theliterature by different designations. It is now universally recognizedthat viruses previously denominated lymphadenopathy associated virus(LAV), immune deficiency associated virus (IDAV1 and IDAV2), humanT-lymphotropic virus type III (HTLV-III), and AIDS related virus (ARV)are all variants of the same retrovirus. See, e.g., Nature, 313:636-637(1985).

A subcommittee empowered by the International Committee on the Taxonomyof Viruses recently proposed that the AIDS retroviruses be officiallydesignated as the "Human Immunonodeficiency Viruses", to be known inabbreviated form as "HIV". Isolates of human retroviruses with clear butlimited relationship to isolates of HIV (for example, more than 20% butless than 50% nucleic acid sequence identity) are not to be called HIVunless there are compelling biological and structural similiarities toexisting members of the group. Science, 232:697 (1986).

Another pathogenic human retrovirus, termed HIV-2 (formerly LAV-2), wasrecently recovered from West African patients with AIDS. Clavel et al.,Science, 233:343-346 (1986). HIV-2 infection is associated with animmunodeficiency syndrome clinically indistinguishable from that causedby the prototype AIDS virus, HIV-1. HIV-2 is related to but distinctfrom HIV-1. Guyeder et al., Nature, 326:662-669 (1987).

Retroviruses genetically related and biologically similar to HIV havebeen isolated from subhuman primates. These retroviruses are designatedas immunodeficiency viruses of the appropriate host species, such as,simian immunodeficiency virus (SIV). SIV was first isolated from captiverhesus macaques (Macaca mulatto) at the New England Regional PrimateResearch Center (NERPRC). This was soon followed by a report ofisolation of an SIV called STLV-III from African green monkeys.Extensive serologic cross-reactivity exists between HIV-2 and SIV.

Transmission of HIV frequently takes place through sexual contact,although people using narcotics intravenously also represent a high-riskgroup. A large number of individuals have also been infected with HIVafter receiving contaminated blood or blood products.

It is becoming more evident that other factors are important in thetransmission of HIV in view of the occurence of AIDS in areas endemic ofmalaria. One factor felt etiologic in AIDS is defective Fc-receptorfunction in the reticuloendothelial system. B. S. Bender, J. Infect.Dis. , 152:409-412 (1985). Defective Fc-receptor function on red bloodcells and subsequent clearance of these IgG coated red cells has beendemonstrated in AIDS patients. In addition, retrovirus antibodyreactivity to the AIDS virus has shown considerable cross-reactivitywith antibody levels against Plasmodium falciparum. R. J. Biggar et al.,Lancet, Sep. 7, 1985:520-523. While there remains some debate about thecross-reactivity, R. J. Biggar et al., New Engl. J. Med., 315:57-8(1986), A. E. Greenburg et al., Lancet, Aug. 2, 1986:247-249, thisdiscussion does not take into account the possibility of dual exposureand clearance of the infective agent of AIDS accompanied by developmentof antibodies to the AIDS virus. The antibody cross-rectivity betweenAIDS and malaria is further exhibited by the effectiveness of a ratherspecific antimalarial agent in opportunistic infections.Pyrimethamine-sulfadoxine in combination has been shown effective in thetreatment of Pneumocystis carinii infections in patients with AIDS. R.D. Pearson et al., Ann. Int. Med., 106:714-718 (1987).

The existence of multiple human immunodeficiency viruses, such as HIV-1and HIV-2, presents a complex epidemiologic picture. There is a commonbelief that an effective vaccine or pharmaceutical composition againstHIV infection must be developed in order to stem the spread of theseretroviruses. Work is progressing on the development of a vaccine, butan effective agent has not yet been found. Thus, there exists a need inthe art for a method of inhibiting the activity of HIV in vivo.

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art. Moreparticularly, this invention provides a method for inhibiting theactivity of human immunodeficiency virus (HIV) in vivo, wherein themethod comprises administering to a human host an antimalarial drugwhich is capable of exhibiting a protective effect, a curative effect,or of preventing transmission of malaria in humans. The antimalarialdrug is selected from the group consisting of

(a) alkaloids;

(b) 9-amino-acridines;

(c) 4-aminoquinolines;

(d) 8-aminoquinolines;

(e) biguanides;

(f) dihydrofolate reductase inhibitors;

(g) sulfones;

(h) sulfonamides;

(i) mefloquine;

(j) halofantrine;

(k) hydroxyanilio-benzo-naphtyridines; and

(l) sesquiterpene lactones;

The antimalarial drug is administered to the human in an amountsufficient to prevent or at least inhibit infection of T lymphocytes byHIV in vivo or to prevent or at least inhibit replication of HIV invivo.

The evasiveness and diversity of HIV has made a definitive treatmentdifficult. Presented here is an agent and a method capable of preventingthe spread or acquisition of HIV infection and of assisting in thetreatment of such infection.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The method of this invention is useful for the treatment of humanseither infected with HIV or susceptible to HIV infection. The expression"HIV infection" includes all of the types of infections included in theclassification system published by the Centers for Disease Control,Atlanta, Ga. These infections include acute HIV infection; asymptomaticconditions; persistent generalized lymphadenopathy; and other diseases,such as constitutional disease, neurological disease, secondaryinfections, secondary cancers, and other conditions attributed to HIVinfection or immunosuppression.

The method of the invention is especially useful for inhibiting theactivity of HIV-1 or HIV-2 in humans infected therewith. While thisinvention is described with reference to human isolates identified inthe literature as LAV-I, HTLV-III, and LAV-II, it will be understoodthat this invention extends to the same or equivalent retroviruses.These retroviruses are believed to be the causative agents of AIDS andHIV disease.

For the purpose of this disclosure, a virus is considered to be the sameas or equivalent to LAV/HTLV-III if it substantially fulfills thefollowing criteria:

(a) The virus is tropic for T-lymphocytes, especially T-helper cells(CD4⁺);

(b) The virus is cytopathic for infected CD4⁺ cells;

(c) The virus encodes an RNA-dependent DNA polymerase (reversetransciptase) which is Mg2⁺ -dependent and can employ oligo(dT)₁₂₋₁₈ asa primer for reverse transcription from its 3' LTR;

(d) The virus is substantially cross-reactive immunologically with theproteins encoded by the gag and env regions of LAV/HTLV-III; and

(e) The virus shares substantial nucleotide homology (approximately75-100%) and amino acid sequence homology (approximately 75-100%) withLAV or HTLV-III.

The principal pharmaceutically active ingredient employed in the methodof the invention is an antimalarial drug. As used herein, the term"antimalarial drug" includes drugs that have a protective (prophylactic)effect against malarial infection, a curative (therapeutic) effectagainst malarial infection, or prevent transmission of malaria inhumans. Thus, antimalarial drugs include drugs that are used beforemalaria infection occurs or before malaria infection becomes evidentwith the aim of preventing either the occurence of the infection or anyof its symptoms. Antimalarial drugs also include drugs that aretherapeutically used for their action on established malaria infection.Further, antimalarial drugs include drugs that are useful for theprevention of infection of humans by malarial vectors, including drugsthat intervene or interfere with the malaria parasite life cycle in ahuman host.

Antimalarial drugs that can be employed in practicing the method of thisinvention are selected from the group consisting of the alkaloids, suchas quinine and quinidine; 9-amino-acridines, such as mepacrine;4-aminoquinolines, such as chloroquine and amodiaquin; and the8-aminoquinolines, such as primaquine, pamaquine, and quinocide.Biguanides, such as proguanil (chlorguanide) can also be employed.Examples of other suitable antimalarial drugs are diaminopyrimidines,such as pyrimethamine, or other dihydrofolate reductase inhibitors, suchas trimethoprim; sulfones, such as diaminodiphenyl sulfone (DDS ordapsone) and diformyl diphenyl sulfone; and sulfonamides, such assulfadiazine, sulfamethoxypyrazine, sulfamonomethoxine, sulfadoxine, andsulfamethoxazole. Mefloquine; halofantrine;hydroxyanilino-benzo-naphtyridines, such as pyronaridine; andsesquiterpene lactones, such as artemisinine, can also be employed.These antimalarial drugs and methods of preparing the drugs are known inthe art.

Combinations of one or more of the antimalarial drugs, other thanpyrimethamine-sulfadoxine, can be employed in practicing the method ofthis invention. Thus, for example, pyrimethamine and chlorquine can beemployed in combination. Similarly, amodiaquine can be employed togetherwith primaquine, primethamine can be combined with dapsone, chloroquinecan be employed with primaguanine or chlorproguanil, pyrimethamine orother antifolate drugs can be combined with sulfadoxine, andpyrimethamine can be employed together with sulfalene or sulfadiazine orsulfamethoxypyrazine.

The antibiotics used as antimalarial drugs have not been found to beeffective when used alone-in the method of the invention. Thus, theexpression "antimalarial drug" as used herein is intended to excludeantimalarial antibiotics when used alone in practicing the method of theinvention. Nevertheless, it will be understood that antimalarialantibiotics, such as tetracycline and clindamycin (doxycycline,lincomycin), can be employed in combination with one or more of theaforementioned antimalarial drugs.

It will also be understood that the method of this invention can bepracticed with compounds that change in vivo into one of theaforementioned antimalarial drugs, as well as compounds that producemetabolites in vivo similar to the metabolites formed from theaforementioned antimalarial drugs.

The antimalarial drugs can be employed in the form of the free base orin the form of a pharmaceutically acceptable acid addition salt.Examples of suitable salts are the chlorides, hydrochlorides, sulfates,phosphates, and diphosphates. Other water soluble, non-toxic, inorganicand organic salts can also be employed.

In practicing the method of the invention, the antimalarial drug isadministered to a human host using one of the modes of administrationcommonly employed for administering the drug as an antimalarial agent tohumans. Thus, for example, the drug can be administered to the host bythe oral route or parenterally, such as by intravenous or intramuscularinjection. For purposes of injection the compounds described above canbe prepared in the form of solutions, suspensions, or emulsions invehicles conventionally employed for this purpose. Other modes ofadministration can also be employed.

The antimalarial drug is employed in the method of the invention in anamount sufficient to provide an adequate concentration of the drug toprevent or at least inhibit infection of T lymphocytes by HIV in vivo orto prevent or at least inhibit replication of HIV in vivo. The amount ofthe drug thus depends upon absorption, distribution, and clearance bythe human host. Of course, the effectiveness of the antimalarial drugsis dose related. The dosage of the antimalarial drug should besufficient to produce a minimal detectable effect, but the dosage shouldbe less than the established lethal dose. The dosage of the antimalarialdrug administered to the host can be varied over wide limits. Thecompounds can be administered in the minimum quantity which istherapeutically effective and the dosage can be increased as desired upthe maximum dosage tolerated by the patient. The antimalarial drug canbe administered as a relatively high loading dose, followed by lowermaintenance dose, or the drug can be administered in uniform dosages.

The dosage and the frequency of administration will vary with theantimalarial drug employed in the method of the invention. Theantimalarial drugs can be employed in this invention in an amount of upto about 6 times the recommended maximum dosage for the treatment ofmalaria. Generally, the dosage will not exceed about 5 times therecommended maximum dosage for the treatment of malaria, and most oftennot more than about 4 times such recommended maximum dosage. Followingare examples of suitable dosage levels.

Quinine dihydrochloride or sulfate can be orally administered in tabletscontaining 300-650 mg of the drug, 2-3 tablets daily. Liquidformulations of this drug can be administered by injection usingsolutions containing 300-500 mg/ml of the drug over a period of about3-4 hours until 600 mg are administered, and the procedure can berepeated 2-3 times in 24 hours.

The antimalarial drugs chloroquine phosphate and chloroquine sulfate canbe orally administered in tablet form (100 mg, 150 mg, and 300 mg) at300 mg-600 mg over one week. Solutions of these antimalarial drugs canbe intramuscularly or intravenously injected as 5% solutions at a dosageof 200-300 mg intramuscularly repeated in six hours or intravenously(drip) 300-600 mg in 500 ml saline.

Amodiaquaine hydrochloride or free base can be orally administered intablets containing 200 mg of the drug at a rate of 400-2,000 mg over oneweek. As another example, primaquine diphosphate can be orallyadministered in tablets containing 5 mg-7.5 mg of the drug at a rate of2-3 tablets per day.

Proguanil can be orally administered in tablets of 100 mg each at a rateof 1-2 tablets daily. Chlorproguanil can be orally administered intablets containing 20 mg of the drug at a rate of 1 tablet per week.Similarly, tablets containing pyrimethamine (25 mg) can be administeredat a rate of 1 tablet per week.

Mefloquine is also suitable for-oral administration. Tablets containing250 mg of the drug can be administered as a single or divided dose of1,500 mg.

The dose of the antimalarial drug is specified in relation to an adultof average size. Thus, it will be understood that the dosage can headjusted by 20-25% for patients with a lighter or heavier build.Similarly, the dosage for a child can be adjusted using well knowndosage calculation formulas.

Primaquine through its more effective extraerythrocytic effect on thereticuloendothelial system and shedding of the virus is a preferredantimalarial drug for use in the method of the invention. The dose foradults is about 15 mg/day base (26 mg/day salt) orally or about 45 mg/wkbase (79 mg/wk salt) orally. For children the dose is about 0.3 mg/kgper day base (0.5 mg/kg per day salt) orally or about 0.9 mg/kg per weekbase (1.5 mg/kg per week salt) orally.

As previously described, any of the antimalarial drugs can be employedin this invention at dosage levels that are multiples of the recommendeddosage for the treatment of malaria. Thus, for example, the adult doseof primaquine can be up to about 250 mg/wk base. As another example,chloroquine can be employed in this invention at adult dosage levels ofabout 1800 mg/wk.

Because of the risk of hemolysis, particularly in individuals withglucose-6-P-dehydrogenase deficiency, other alternatives exist. Theseinclude the less toxic 8-aminoquinolines, or other carrier linkedprimaquine agents. J. Hofsteenge et al., J. Med. Chem., 29:1765-1769(1986).

There is much speculation as to mechanism of action of the antimalarialdrugs in the prevention and treatment of malaria. D. A. A. Akintonwa, J.Theor. Biol., 106:78-87 (1984). R. Deslauriers et al., Biochimica etBiophysica Acta, 931:267-275 (1987). Proposed mechanisms includedinhibition of lysosomal cathepsins, protonation within acidicintracellular compartments, stabilization of membranes, and enzymeinduction. The net effect of antimalarial drugs in the prevention andtreatment of malaria appears to be in the inhibition of sporozoiteinvasion of reticuloendothelial cells. A. L. Schwartz et al., Molec. andBiochem. Parasit. 14:305-311 (1985). In any event, the mechanism bywhich the antimalarial drugs inhibit the activity of HIV in vivo by themethod of this invention is not entirely understood. In fact, themechanism may be due to one factor or to a combination of factors, suchas a modification of one or more genotypic or phenotypic traits of theretrovirus or modification of viral or cellular processes. Thus, theantimalarial drug may inhibit infection of susceptible CD4⁺ cells byinterfering with cell receptor function or the viral envelope receptoror by altering the nature of the binding mechanism. There is evidencethat the retrovirus is translocated into the cell. Thus, theantimalarial drug may alter the translocation process. Further, thegenomic elements that control the expression of the products requiredfor viral replication may be altered or their functioning may beaffected by the antimalarial drugs. Thus, for example, the antimalarialdrugs may function as reverse transcriptase inhibitors or alter thenature of the envelope glycoprotein of the retrovirus or inhibit tatgene expression or affect the level of expression of the env genedeterminants on the cell surface. The antimalarial drugs may alsofunction by mediating the functioning of the reticuloendothelial system,either before or after HIV infection has occurred. For example, theantimalarial drugs may enhance Fc-receptor expression or function invivo in the mononuclear phagocyte system. This may result in a loweredsusceptibility to opportunistic infections associated with AIDS. Theantimalarial drugs may also inhibit proliferation of HIV in infectedphagocytic cells. It will be understood that the antimalarial drugs mayfunction in any of these ways or by combinations thereof or by otherways not currently recognized. In any event, the antimalarial drugs canbe administered to the patient in sufficient amounts to achieve one ormore of these effects.

The effectiveness of the antimalarial drugs in preventing or inhibitinginfection of cells is demonstrated using standard in vitro assays. Thus,the inhibitory effect of the antimalarial drugs on HIV infection orreplication can be demonstrated by cultivating the virus orvirus-infected cells in the presence and in the absence of theantimalarial drug and then comparing the results.

One method involves cultivation of normal peripheral blood mononuclearcells or other cultured cells exposed to the virus. The test forinfectivity can use either free virus or virus-infected cells that areco-cultivated with a sensitive indicator cell. The virus replicates inthe recipient indicator cell. By comparing viral replication in theabsense of the antimalarial drug with viral replication in the presenceof the drug, the inhibiting effect of the drug on HIV can bedemonstrated.

Viral replication can be determined by monitoring reverse transcriptase(RT) activity. Virus production can also be determined by electronmicroscopic evidence of virus particles and various viral proteins.Various detection protocols for viral proteins make use of standardantibodies prepared against the virus in many animal species.Immunofluorescence can detect viral proteins rapidly and sensitivehyperimmune sera can be prepared. Radioimmune and ELISA assays can alsobe used to measure presence of viral proteins by competition in a systemwhere a radiolabeled or colorimetrically identifiable antigen-antibodysystem can be perturbed by the addition of reactive antigen.

Another approach for demonstrating the effectiveness of the antimalarialdrugs involves detection of the virus by detecting unintegrated andintegrated viral DNA as well as viral mRNA. Nature, 312:166-169 (1984).Southern and Northern blot hybridization techniques are useful indetermination of the relative amounts of viral DNA and RNA of thevirus-harboring cells and tissues. Science, 227:177-182 (1985). A probecan be constructed for integrated provirus using molecularly clonedlabeled proviral DNA, and then one can determine in a DNA transferexperiment whether there is a proviral genome integrated in cellular DNAby hybridization. If only a few cells contain the provirus, in situhybridization can be attempted.

More particularly, in a typical experiment, cells are exposed tocell-free virions at a multiplicity of viral particles per cell andcultured in the presence or absence of antimalarial drugs. Highmolecular weight DNA is extracted at various times and assayed for itscontent of viral DNA using a radiolabelled HIV probe. Nature,312:166-169 (1984). In the absence of antimalarial drugs under theculture conditions, viral DNA is detected. In contrast, in DNA fromcells that have been completely protected by antimalarial drugs, neitherunintegrated nor integrated DNA is detected.

It is also possible to monitor viral replication by determining whetherviral RNA is expressed in target T cells exposed to the virus andcultured with or without antimalarial drugs. In this experiment, cellsare exposed to HIV, and RNA is extracted from the cells. Extracted RNAis then assayed for the content of viral mRNA by Northern blothybridization using a radiolabelled HIV antisense RNA probe. Science,227:177-182 (1985). In the absence of dideoxynucleosides, viral mRNA isdetectable. When these cultures are maintained for extensive periods oftime in the presence of antimalarial drugs, viral RNA expression in thecells is not detected, or if detected, is present in substantiallyreduced amount. This assay system makes it possible to assess thecapacity of the antimalarial drugs to inhibit HIV nucleotide synthesisand mRNA expression in T cells exposed to the virus.

In addition, HIV-I-infected cell lines MT2 and MT4 are sensitiveto thecytopathic effect of the virus. Thus, these cells can be used inplaque-forming assays to demonstrate the inhibitory effect of theantimalarial drugs on HIV infection or prolifertion. Science,229:563-566 (1985).

The effectiveness of the antimalarial drugs in inhibiting HIV infectionor HIV replication is also demonstrated in vitro by determining theinhibitory effect of the drugs on viral p24 gag protein expression in H9cells, partially resistant to the cytopathic effect of HIV. M. Popovicet al, Science, 224:497-500 (1984). Following exposure of H9 cells tothe virus, p24 antigen is determined by indirect immunofluorescenceassay using murine monoclonal antibody. H9 cells are relativelyresistant to the cytopathic effect of HIV, and p24 Sm protein expressionfollowing exposure to HIV can be used as an index of viral infectivityand replication in vitro. It is then possible to assess antiviral effectof the compounds by quantitating the HIV p24 gag protein expression inH9 cells that were exposed to the virus but cultured with drugs. Proc.Natl. Aca. Sci. USA, 82:4813-4817 (1985).

Anti-HIV activity can also be demonstrated by showing the inhibitoryeffect of the antimalarial drugs on the cytopathic effect of HIV. Animmortalized helper-inducer T-cell clone (ATH8) has been described andshows the cytopathic effect of the virus. When ATH8 cells are culturedin a test tube, these cells form a pellet at the bottom of the tube andthe pellet size reflects the number of viable target T cells. Underthese conditions, the cytopathic effect is amenable to direct visualinspection. When exposed to HIV in the absence of the protectiveantimalarial drugs, the virus exerts a profound cytopathic effect on thetarget T cells. When the antimalarial drug is added in culture at thebeginning of the assay, target ATH8 cells are protected against thecytopathic effect of the virus, and can continue to grow. Theseobservations can be substantiated and quantitated by counting the numberof viable cells with a dye exclusion method. This HIV cytopathic effectinhibition assay thus permits the simultaneous assessment of potentialantiviral activity and toxicity of selective compounds. See, M. Hiroakiet al, Science, 240:646-649 (1988), for additional details of thisassay.

Finally, the activity of the antimalarial drugs can be shown by normal Tcells -in vitro. In this assay, normal cloned helper-inducer T cells,such as normal tetanus toxid-specific helper/inducer clonal T cells(TMII cells), are used to monitor the effect of the drugs onantigen-induced proliferative response. Details of this technique aredescribed by H. Mitsuya et al., Science, 240:646-649 (1988).

The effectiveness of the antimalarial drugs in preventing or inhibitingHIV infection or replication can be confirmed in vivo with thechimpanzee. Persistent infection with HIV is demonstrated with thisprimate, although AIDS-like disease is not shown. The effectiveness ofthe antimalarial drugs can be demonstrated by comparing treated anduntreated chimpanzees for seroconversion to HIV antigens, by reisolatinginfectious virus from the animals, by demonstrable lymphadenopathy, byalterations in T4 or TS lymphocyte levels, or by combinations of thesefactors.

HIV required to carry out these assays can be obtained from conventionalsources using conventional techniques. For instance, mononuclear cellsprepared from peripheral blood, bone marrow, and other tissues frompatients and donors can be stimulated with mitogen(phytohemagglutinin-P) for 48 to 72 hrs and established in cell cultureusing growth medium supplemented with T cell growth factor (TCGF).,Virus can be detected by:

(1) monitoring supernatant fluids for viral reverse transcriptaseactivity; (2) transmitting virus to fresh normal human T-lymphocytes(e.g., umbilical cord blood, adult peripheral blood, or bone marrowleukocytes) or to established T-cell lines; M. Popovic et al, Science,224:497 (1984); (3) electron microscopic observation of fixed andsectioned cells; and (4) testing for antigen expression by indirectimmunofluorescence or Western blot procedures using serum fromseropositive donors. The detection of virus-positive cells and thecharacterization and comparison of viral isolates can be conducted usingHIV-specific immunologic reagents and nucleic acid probes. F.Barre-Sinoussi et al., Science, 220:868-871 (1983); R. C. Gallo et al.,Science 220:865-867 (1983).

Many established cell lines have been tested as possible targets forHIV-1 infection. One neoplastic aneuploid T-cell line derived from anadult with lymphoid leukemia and termed HT is susceptible to infectionwith HTLV-III. HT cells continuously produce HTLV-III after parentalcells are repeatedly exposed to concentrated cell culture fluidsharvested from short-term cultured T-cells (grown in TCGF) thatoriginated from patients with lymphadenopathy syndrome or AIDS. Whencell proliferation declines, usually 10 to 20 days after exposure to theculture fluids, the fresh (uninfected) HT parental cells are added tocultures. To improve permissiveness for HIV and to preserve permanentgrowth and continuous production of virus, the HT cell population hasbeen extensively cloned. Several of these clones of HT cells have beenmaintained in cell culture. In addition to HT, there are several other Tor pre-T human cell lines that can be infected by and continue toproduce HIV. Examples of these cell lines are H4, H9, HUT 78, CEM, Molt3, and Ti7.4. Gallo et al., U.S. Pat. No. 4,652,599. Furthermore, someB-lymphoblastic cell lines can also be productively infected by HIV. L.Montagnier et al., Science, 225:63-66 (1984).

The antimalarial drugs and their pharmaceutically acceptable salts canbe used in mammalian, including but not limited to human, therapy in theform of pills, tablets, lozenges, troches, capsules, suppositories,injectable or ingestable solutions and the like in the treatment ofcytopatic and pathological conditions in humans and susceptiblenon-human primates caused by disturbances in the functioning of theimmune systems, in particular as regards depression in the level of CD4⁺T lymphocytes.

Appropriate pharmaceutically acceptable carriers, diluents, andadjuvants can be combined with the antimalarial compounds describedherein in order to prepare the pharmaceutical compositions for use inthe treatment of pathological conditions in mammals. The pharmaceuticalcompositions of this invention contain the active compound together witha solid or liquid pharmaceutically acceptable nontoxic carrier. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable, or synthetic origin.Examples of suitable liquids are peanut oil, soybean oil, mineral oil,sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Physiologicalsaline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatine, malt, rice, flour, chalk, silica gel, magnesiumcarbonate, magnesium stearate, sodium stearate, glycerol monstearate,talc, sodium chloride, dried skim milk, glycerol, propylene glycol,water, ethanol, and the like. These compositions can take the form ofsolutions, suspensions, tablets, pills, capsules, powders,sustained-release formulations and the like. Suitable pharmaceuticalcarriers are described in "Remington's Pharmaceutical Sciences" by E. W.Martin. The pharmaceutical compositions contain an effective therapeuticamount of the active compound together with a suitable amount of carrierso as to provide the form for proper administration to the host.

In summary, the antimalarial drugs are especially useful as antiviralagents for the therapeutic treatment of humans. They exhibit potentantiviral activity against the AIDS viruses, which is highly unusual andunexpected in view of the very limited and specific antiviral activityof the prior art antiviral agents. The antimalarial drugs exhibit markedsuppression of HIV multiplication and virus-induced cell injury inanimal and human cell tissue culture systems. The antimalarial drugs canreduce mortality and morbidity manifestations in humans, including areduction in the occurrence of opportunistic infections associated withAIDS and a reduction in progressive, degenerative effects of HIV on thecentral nervous system.

What is claimed is:
 1. A method for preventing or inhibiting theactivity of human immunodeficiency virus (HIV) in vivo, wherein themethod comprises administering a 4-aminoquinoline or a pharmaceuticalsalt thereof to a human in need thereof, in an amount sufficient toprevent or inhibit infection of T-lymphocytes by HIV or to prevent orinhibit replication of HIV in vivo.
 2. The method of claim 1, whereinthe 4-aminoquinoline is administered to the human in an admixture with apharmaceutically acceptable carrier.
 3. The method of claim 1, whereinthe 4-aminoquinoline is selected from the group consisting ofchloroquine, chloroquine phosphate, chloroquine sulphate, andamodiaquin.
 4. The method of claim 1, wherein the 4-aminoquinoline ischloroquine.
 5. The method of claim 1, wherein the 4-aminoquinoline ischloroquine phosphate.
 6. The method of claim 1, wherein the4-aminoquinoline is chloroquine sulphate.
 7. The method of claim 1,wherein the 4-aminoquinoline is amodiaquin.